Chapter 2 THYMIC EPITHELIAL CELLS REQUIRE VPS34 TO MAINTAIN CELLULAR
2.4 Materials and methods
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80 C57BL/6-Tg(TcraTcrb)1100Mjb/J
(OTI)/
C57BL/6J
The Jackson Laboratory
03831 B6.Cg-Tcratm1Mom
Tg(TcrLCMV)327Sdz/TacMmjax (P14)/
C57BL/6J
The Jackson Laboratory
37294
B6.Cg-Tg(TcraTcrb)425Cbn/J (OTII)/
C57BL/6J
The Jackson Laboratory
04194 Tg(TcraLLO56,TcrbLLO56)#Pmal
(LLO56)
Dr. Paul Allen, Washington University School of
Medicine
(Milam and Allen, 2015)
Tg(TcraLLO118,TcrbLLO118)#Pmal (LLO118)
Dr. Paul Allen, Washington University School of
Medicine
(Milam and Allen, 2015)
Tg(TcraH-Y,TcrbH-Y)1Pas (Marilyn)
Dr. Maria-Luisa Alegre, University of
Chicago
(Lantz et al., 2000) C57BL/6-Tg(CAG-
RFP/EGFP/Map1lc3b)1Hill/J (CAG-RFP-EGFP-LC3)/
C57BL/6J
The Jackson Laboratory
027139
B6.129S-Atg5tm1Myok (Atg5f/f)/
C57BL/6J
Dr. Akiko Iwasaki, Yale University School of Medicine
(Hara et al., 2006) C57BL/6-Foxp3tm1Flv/J
(Foxp3-RFP)/
C57BL/6J
The Jackson Laboratory
008374 B6;129S4-H2-DMatm1Luc/J
(H2-DMa-/-)/
C57BL/6J
Dr. Paul Allen, Washington University School of
Medicine
(Martin et al., 1996)
B6;129S6-Gt(ROSA)26Sortm14(CAG-
tdTomato)Hze/J (Ai14)/
C57BL/6J
Dr. Mark Anderson, University of California San Francisco Medical
Center
(Madisen et al., 2010)
B6.SJL-Ptprca Pepcb/BoyJ (CD45.1)/
C57BL/6J congenic strain
The Jackson Laboratory
006785
81 B6.129S7-Rag1tm1Mom/J
(RAG-/-)/
C57BL/6J
The Jackson Laboratory
002014
Flow cytometric analysis
T cells from thymi, lymph nodes, and spleens were obtained by mashing the organ through a 70 µm cell strainer (Fisher Scientific International, Inc, 22-363-548) and suspended in FACS buffer (PBS containing 2% fetal bovine serum and 0.05% sodium azide) for further processing as outlined below.
For T cell analysis, cells were incubated with conjugated monoclonal antibodies against mouse CD4 (Tonbo), CD8 alpha (Tonbo), CD3 epsilon (BioLegend), CD44 (Tonbo), CD62L (BD Biosciences), CD69 (BD Biosciences), TCR beta (Tonbo), CD24 (BD Biosciences), CD25 (Tonbo), CD5 (BioLegend), TCR V alpha 2 (BioLegend), TCR V beta 5.1 (BioLegend), and/or TCR V beta 6 (BD Biosciences). For intracellular staining, cells were processed and stained for extracellular markers as described prior to fixation/permeabilization overnight using the Foxp3 transcription factor staining buffer kit (Tonbo) according to the manufacturer’s protocol. Cells were then incubated with conjugated monoclonal antibody against Foxp3 (Tonbo).
For TEC analysis, thymi were dissected and digested in 1 mg/ml collagenase/dispase (Roche, Basel, Switzerland) and passed through a 100 µm mesh to remove debris. Monoclonal antibodies against mouse CD45.2 (Tonbo), EpCam (BioLegend), Ly51 (eBioscience), CD80 (BD Biosciences), Kb (BD Biosciences), CLIP- bound-I-Ab (clone 15G4, Santa Cruz), IgG1 (eBioscience) and I-A/I-E (eBioscience), and the UEA1 lectin (Vector Labs) were used in the TEC analysis. For intracellular staining, cells were processed and stained for extracellular markers as described prior to
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fixation/permeabilization overnight using the Foxp3 transcription factor staining buffer kit (Tonbo) according to the manufacturer’s protocol. Cells were then incubated with conjugated monoclonal antibody against Ki67 (Biolegend).
Flow cytometric analyses were performed using a Canto II or 4 laser Fortessa (BD Biosciences) flow cytometer. The acquired data were analyzed using FlowJo software (Version 10.0.7, Treestar, Palo Alto, CA).
Fluorescence activated cell sorting
Cells were stained as described for flow cytometry. ~1.1 x 105 TCRβhi, CD4+CD8- thymocytes were sorted into cold HEPES buffer (PBS with 2% FBS and 0.025 M HEPES) and immediately used for genomic DNA (gDNA) isolation. Sorting was performed using a FACS Aria III (BD Biosciences) flow cytometer.
Immunofluorescence and histology
For cryosectioning, thymi were snap-frozen on dry ice and stored at −80°C. Tissues were sectioned at 8 µm thickness and fixed in ice-cold acetone for 2 min. Tissues were rinsed with PBS, blocked with 10% donkey serum in PBS for 30 min at room temperature, then incubated with the primary antibody anti-Keratin 5 (abcam) or biotin-labeled UEA-1 lectin (Vector Labs) overnight at 4°C. Secondary detection was performed with goat anti-rabbit IgG (Jackson ImmunoResearch) and Streptavidin-Cy3 (Vector Labs). Sections were examined by fluorescent microscopy using a Zeiss LSM 710 confocal microscope.
For paraffin sectioning, tissues were collected and fixed in 10% PFA for formalin overnight. Tissues were dehydrated through an ethanol series and embedded in paraffin
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wax using standard procedures. Sections (8 mm) were cut and rinsed in xylene before rehydration through a reverse ethanol series. Hematoxylin and eosin staining was performed on paraffin sections using standard procedures; sections were then imaged on a Nikon AZ 100M widefield microscope.
Thymus grafts
Thymic lobes were dissected from newborn Rosa26-CreERT2 mice and placed in ice cold PBS. Thymi were separated into single lobes and connective tissue removed. A single lobe was transplanted under the kidney capsule of each 6-week-old male C57BL/6 recipient, as described (Jain et al., 2017). Grafts were allowed to reconstitute for 3 weeks, then mice were orally gavaged with 3 mg of tamoxifen (Sigma-Aldrich) in peanut oil for three alternate days and analyzed 3 weeks after the final dose.
TCR sequencing and analysis Sample preparation
Genomic DNA was prepared from CD4+CD8- TCRβhi thymocytes sorted from 4- or 6-day- old mice using a standard Qiagen® DNA extraction kit according to the manufacturer’s instructions. Library preparation and sequencing sample data were generated using the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle, WA). The somatically rearranged mouse TCRβ CDR3 regions were amplified from genomic DNA using a two- step, amplification bias-controlled multiplex PCR approach. The first PCR consisted of forward and reverse amplification primers specific for every known V and J gene segment and amplified the hypervariable CDR3 of the immune receptor locus. The second PCR
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added a proprietary barcode sequence and Illumina® adapter sequences. In addition, reference gene primers were included in the PCR reaction to quantify total sequenceable nucleated cells and to accurately measure the fraction of T cells in each sample. CDR3 and reference gene libraries were sequenced on an Illumina® instrument according to the manufacturer’s instructions.
Data analysis
Raw sequence reads were demultiplexed according to Adaptive’s proprietary barcode sequences. Demultiplexed reads were then further processed to remove adapter and primer sequences, and to identify and remove primer dimers, germline, and other contaminant sequences. The filtered data were clustered using both the relative frequency ratio between similar clones and a modified nearest-neighbor algorithm, to merge closely related sequences to correct for technical errors introduced through PCR and sequencing. The resulting sequences were sufficient to allow annotation of the V, D, and J genes and the N1 and N2 regions constituting each unique CDR3 and the translation of the encoded CDR3 amino acid sequence. Gene definitions were based on annotation in accordance with the IMGT database (www.imgt.org). The set of observed biological TCRβ CDR3 sequences was normalized to correct for residual multiplex PCR amplification bias and quantified against a set of synthetic TCRβ CDR3 sequence analogues. Data were analyzed using the immunoSEQ Analyzer toolset. Data were imported into GraphPad Prism 6.0 for statistical analyses and graph generation.
85 Induction and evaluation of active EAE
Six- to eight-week-old animals were used for EAE induction, as described (Miller et al., 2010). Under isoflurane anesthesia, the animals were subcutaneously injected at two sites into the flank with 200 µl of 1 mg/ml MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) peptide (Biomatik) emulsified in Freund’s complete adjuvant [2 mg/ml Mycobacterium tuberculosis extract H37Ra in incomplete Freund’s adjuvant (BD bioscience, 263910)].
Immediately after immunization and 48 h later, all mice received 400 ng of pertussis toxin (Calbiochem, 516560) by i.p. injection. Mice were scored daily in a blinded manner according to the following scale: 0, no clinical signs; 0.5, partially limp tail; 1, paralyzed tail; 1.5, paralyzed tail and hind leg inhibition; 2, loss in coordinated movement, hind limb paresis; 2.5, one hind limb paralyzed; 3; both hind limbs paralyzed; 3.5, hind limbs paralyzed, weakness in forelimbs; 4, forelimbs paralyzed; 5, moribund.
TEC culture
C9 cells (Wertheimer et al., 2018) (kindly provided by Dr. M. van den Brink from Memorial Sloan Kettering Cancer Center) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone Fetal Clone III), 100 units per ml penicillin, and 100 mg/ml streptomycin.
CRISPR-Cas9 editing of Vps34 in C9 cells
For excision of the Vps34 gene, we used a lentivirus harboring either lentiCRISPR v2 empty vector (Addgene, #52961) or lentiCRISPR v2 sgVps34. We amplified lentiviruses as described (Song et al., 2014). Parental C9 cells were transfected with the respective
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virus to produce control sgLacZ cells and stable Vps34-deficient sgVps34 cells. gRNA sequences for Vps34 were TCTCGTAGCATGTTTCGCCA. Efficient gene deletion was confirmed by western analysis.
Western blotting
C9 cells were lysed in RIPA buffer (Sigma Aldrich) containing a cocktail of protease inhibitors (Roche) with brief sonication. Total proteins (50 µg) were separated on SDS- PAGE gels, transferred onto PVDF membranes, and incubated with primary antibodies.
Secondary antibodies were subsequently applied and proteins were detected with ECL substrate (ClarityTM, Bio-Rad) or using an LI-COR Odyssey Infrared Imaging System.
Generation of bone marrow chimeras
Recipient mice were lethally irradiated (1,000 cGy) and 6 hours later injected with
~107 bone marrow cells derived from donor mice. Mice were used for experiments at 5 to 6 weeks after injection of bone marrow cells.
OTII x Rip-mOVA system to test negative selection
Following reconstitution with OTII TCRTg bone marrow, recipient mice (Vps34f/f, Aire- CreERT2 Vps34f/f, Aire-CreERT2 Vps34f/f Rip-mOVA) were treated with tamoxifen suspended in peanut oil (3 mg) by oral gavage every other day for 14 days and then analyzed.
87 Statistical analysis
Statistical analysis was performed with GraphPad Prism 6.0 (GraphPad Software). Data are presented as mean ± S.D. N was at least two for each genotype in each experiment, as indicated in the text and/or figure legends. Comparisons between two groups were made using two-tailed student’s t-test; p < 0.05 was considered significant.
EAE
EAE clinical scores and disease incidence were analyzed using ANOVA and Kaplan- Meier curves by log-rank test. Peak disease score was analyzed by two-tailed student’s t-test. p < 0.05 was considered significant.
TCR sequencing
Clonality, gene usage, and overlap comparisons were analyzed by two-tailed student’s t- test. CDR3 length was analyzed by Chi-squared test (Miqueu et al., 2007). p < 0.05 was considered significant.
88 Chapter 3
VPS34 PROMOTES MATURE B CELL HOMEOSTASIS YET RESTRICTS GERMINAL CENTER FORMATION